LOCUS       sequence-360642-        5164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating 
            peptide-mNeongreen-Erythropoietin fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5164)
  AUTHORS   Eastwood TA, Baker K, Streather BR, Allen N, Wang L, Botchway SW, 
            Brown IR, Hiscock JR, Lennon C, Mulvihill DP
  TITLE     High-yield vesicle-packaged recombinant protein production from E. 
            coli.
  JOURNAL   Cell Rep Methods. 2023 Feb 2;3(2):100396. doi: 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27.
  PUBMED    36936078
REFERENCE   2  (bases 1 to 5164)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep 
            Methods."; date: "2023-02-2"; volume: "3(2):100396. doi"; pages: " 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27"
COMMENT     Sequence Label: pRSFDuet-1_VNp-mNeongreen-Erythropoietin
FEATURES             Location/Qualifiers
     source          1..5164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             432..1139
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             1668..1685
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1701..1745
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1783..1801)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1797..1844
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2074..2889)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2074..2889)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2797..2816
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2890..2981)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2997..3746)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(3007..3024)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3247..3266)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3942..5024)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4986..5005
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5025..5102)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5148..5164,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tggatgtatt catgaaagga ctttcaaagg ccaaggaggg agttgtggca gctgctgaga
      361 aaaccaaaca gggtgtggca gaagcagcag gaaagacaaa agagggtgtt ctcagatctg
      421 gaggaagcgg aatggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
      481 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
      541 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
      601 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
      661 accctgacgg gatgtcgcct ttccaggccg cgatggtaga tggctccgga taccaagtcc
      721 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
      781 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
      841 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
      901 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
      961 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1021 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1081 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagg
     1141 ctagcagatc tggaggaagc ggagcaccgc ctcgtctgat ttgtgatagc cgtgttctgg
     1201 aacgttatct gctggaagca aaagaagcag aaaacattac caccggttgt gcagaacatt
     1261 gtagcctgaa tgaaaatatc accgttccgg ataccaaagt gaacttttat gcatggaaac
     1321 gtatggaagt tggtcagcag gcagttgaag tttggcaagg tctggcactg ctgagcgaag
     1381 cagttctgcg tggtcaggcc ctgctggtta atagcagcca gccgtgggaa ccgctgcaac
     1441 tgcatgttga taaagcagtt agcggtctgc gtagcctgac aaccctgctg cgtgcactgg
     1501 gtgcacagaa agaagcaatt agccctccgg atgcagcaag cgcagcaccg ctgcgtacca
     1561 ttaccgcaga tacctttcgt aaactgtttc gcgtgtatag caatttcctg cgtggtaaac
     1621 tgaaactgta taccggtgaa gcatgtcgta ccggtgatcg tggtacccat catcatcatc
     1681 atcattaact cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
     1741 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
     1801 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa cctcaggcat
     1861 ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga
     1921 cataagcggc tatttaacga ccctgccctg aaccgacgac aagctgacga ccgggtctcc
     1981 gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2041 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     2101 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2161 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2221 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2281 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     2341 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2401 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcggtc
     2461 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     2521 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2581 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2641 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     2701 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     2761 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     2821 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     2881 atggctcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     2941 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggcatgc agcgctcttc
     3001 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggtgtcagc
     3061 tcactcaaaa gcggtaatac ggttatccac agaatcaggg gataaagccg gaaagaacat
     3121 gtgagcaaaa agcaaagcac cggaagaagc caacgccgca ggcgtttttc cataggctcc
     3181 gcccccctga cgagcatcac aaaaatcgac gctcaagcca gaggtggcga aacccgacag
     3241 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3301 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3361 atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3421 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3481 ccaacccggt aagacacgac ttatcgccac tggcagcagc cattggtaac tgatttagag
     3541 gactttgtct tgaagttatg cacctgttaa ggctaaactg aaagaacaga ttttggtgag
     3601 tgcggtcctc caacccactt accttggttc aaagagttgg tagctcagcg aaccttgaga
     3661 aaaccaccgt tggtagcggt ggtttttctt tatttatgag atgatgaatc aatcggtcta
     3721 tcaagtcaac gaacagctat tccgttactc tagatttcag tgcaatttat ctcttcaaat
     3781 gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc
     3841 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     3901 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3961 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4021 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     4081 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     4141 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     4201 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     4261 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     4321 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     4381 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4441 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4501 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4561 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4621 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4681 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4741 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4801 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4861 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4921 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4981 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     5041 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     5101 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac
     5161 tata
//
